Under this project, we have previously found using intravital microcopy imaging in a humanized mouse model of sickle cell disease (SCD) that adherent leukocytes recruited in inflamed venules, played a direct role in VOC by interacting with circulating erythrocytes (RBC). We have determined that RBCs interact specifically with adherent polymorphonuclear neutrophils (PMNs) in vivo. We have identified E-selectin ligand-1 as a major adhesion receptor sending signals that contribute to activate the beta2 integrin Mac-1 specifically at the leading edge of crawling PMNs. Neutrophil Mac-1 captures circulating RBC and thus contribute to VOC. Our preliminary studies suggest that VOC is mediated by a subset of PMNs (senescent phenotype) which we propose to characterize. Other exciting preliminary studies suggest that neural signals regulate the expression of adhesion molecules on venular endothelial cells through circadian adrenergic signals and that these signals can modulate the inflammatory response and the clearance of senescent PMNs. In Specific Aim 1, we will test whether senescent PMNs promote heterotypic interactions and acute VOC in SCD. We will dissect the contribution of PMN aging vs. activation on their ability to promote in vivo heterotypi RBC-WBC interactions following ex vivo aging and stimulation. We will evaluate gain- and loss-of function model systems that promote or reduce senescent PMN clearance in the bone marrow or survival in the circulation. Preliminary data indicate that circadian adrenergic signals are locally delivered by nerves of the sympathetic nervous system (SNS). However, SNS nerves innervate arterioles, but not venules where leukocytes are recruited, raising the intriguing question as to how these signals are transmitted to venular endothelial cells. In Specific Aim 2, we will define how SNS nerves control endothelial cell function in venules. We will track in vivo signal transduction by imaging calcium waves using GCaMP3 mice following direct neural stimulation of the genitofemoral nerve or pericytes by micropipette stimulation in collaboration with Dr. David Spray (Einstein). We will identify genetically the cellular and molecular basis by specific deletion of connexins or beta2 adrenergic receptors (Adrb2fl/fl) in pericytes or endothelial cells, and we will investigate the functional consequences of tissue-specific deletions on circadian leukocyte recruitment in healthy wild-type mice and in SCD mice. In addition to the SNS, our preliminary data also suggest a role for parasympathetic nervous system (PNS) in leukocyte recruitment. We will investigate in Specific Aim 3 the role of PNS (cholinergic) signals in leukocyte adhesion and sickle cell vaso-occlusion. We will explore in collaboration with Dr. Kevin Tracey (Feinstein Institute for Medical Research), the influence of vagal stimulation and nicotinic receptor signaling. We will also evaluate the role of the type 1 muscarinic receptor (Chrm1) using pharmacologic and genetic analyses since our preliminary studies suggest a role for this receptor in leukocyte trafficking. These studies will provide new insight on the mechanisms regulating sickle cell VOC and lead to novel ways to target inflammation.